The invention described herein relates generally to structures and methods for measuring electromagnetic radiation and, more particularly, to structures and methods for simultaneously measuring a plurality of spectral wavelengths present in fluorescence produced when at least one light source excites individual particles moving sequentially through the fluorescence excitation region of a flow cytometer. This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).
The subject invention is an apparatus for receiving and analyzing electromagnetic radiation having a plurality of wavelengths in a timescale on the order of 0.01 to 0.1 ms. In its simplest embodiment, the apparatus includes a polarization interferometer which derives its resolution from a birefringent element capable of being modulated at greater than 50 kHz, in contrast to the usual slow mechanical scan of an interferometer mirror. It is well known that the spectral resolution of an interferometer based spectrometer is related to the maximum path difference in its interferogram. In the subject invention, a varying path difference is provided by the different indices of refraction for radiation polarized along the principal axes of the modulatable birefringent element. That is, if radiation undergoing investigation is either polarized to begin with or is passed through a polarizing element not having its polarization axis parallel to or orthogonal to either of the two principal axes of the birefringent element, this radiation will be resolved into two substantially collinear beams, each having its polarization axis directed along one of the principal axes. Since the index of refraction for radiation polarized along each of these axes is different, the difference being a time-varying function of time determined by the frequency at which the birefringent element is modulated, a time-varying phase shift is introduced between the components traveling through the birefringent element. Introducing this phase shift is equivalent to introducing a path difference in a conventional interferometer. However, since the birefringent element can be rapidly modulated, a rapidly varying phase shift is introduced, thereby effectively performing the introduction of a path difference in a very short timescale. This becomes important when one wishes to investigate transient phenomena which are not repetitive, such as the rapid analysis of emission from single particles in order to make separation decisions based on certain characteristics in a flow cytometer system. The two phase-shifted beams are then mixed after passing through a second polarizer which selects a single polarization for the two substantially collinear, phase-shifted components. An interference pattern characteristic of the spectral distribution of the radiation under investigation is produced.
Polarization modulators are known in the art. However, the phase shifts introduced by such modulators are 1/4-wavelength of the light employed and not the several wavelengths required and taught by the subject invention in order to obtain reasonable resolution.
U.S. Pat. Nos. 4,086,652 and 4,138,727 to Martz disclose a system for analyzing time-varying phenomena of a repetitive nature. The complete interferogram is obtained by dividing the time period of interest into small time intervals and revisiting a particular interval repeatedly until the desired spectroscopic resolution and signal-to-noise ratio are produced by mechanically scanning a mirror of the interferometer. The reason that any useful spectroscopic information is generated results from the repetitive nature of the phenomenon under investigation, since the scan may effectively be continued for as long as desired, thereby permitting arbitrarily good resolution to be obtained, within the characteristics of the optical system.
"Polarised Interferometric Spectrometry for the Millimetre and Submillimetre Spectrum," by D. H. Martin and E. Puplett, Infrared Physics 10, 105 (1969) discloses the use of polarizers for splitting a light beam into two components and for introducing a path difference therein before recombination thereof for the purpose of interferometric spectrometry. No birefringent modulator apparatus is suggested for rapidly performing the equivalent function of providing a variable or scannable path difference between the components. This result is presumably achieved by mechanically moving the polarizers. "Differential Absorption at High Modulation Frequencies Using a Fourier Transform Infrared Spectrometer," by L. A. Nafie and D. W. Vidrine, Multiplex and/or High Throughput Spectroscopy, SPIE 191, 56 (1979), discloses a birefringent element used to modulate linearly polarized light. However, the purpose of so doing is to analyze the linear and circular dichroism of the sample and not to provide a substitute for mechanically sweeping the mirror to obtain a Fourier spectrum.
The present invention is of particular value when used with flow cytometers. Measurements of the fluorescence from fluorochromes (fluorescent dyes) provide quantitative information about the cell components to which the dyes are bound. Flow cytometers can measure cellular properties such as cell size, DNA content, protein content and cell membrane permeability. They can also measure cellular antigens and the shape, size and DNA content of individual chromosomes. The analysis of cells stained simultaneously with several fluorochromes provides the phenotypic information that can be obtained with the particular fluorochromes and allows cell sorting on the basis of this phenotypic description.
Multiple laser flow cytometers have generally used two or three lasers to excite the same number of fluorochromes. In conventional flow cytometers, each fluorochrome produces fluorescence detected by a separate detector. The number of fluorochromes and the number of detectors are usually the same. The number of detectors represents the number of spectral channels of a conventional flow cytometer.
The increasing availability of new monoclonal antibodies which recognize many different antigens, of fluorochromes which specifically bind to various cell components, and of fluorochromes whose emission and/or excitation spectra change as a result of changes in the physiological state of cells make the development of flow cytometers with an increased number of spectral channels highly desirable. The spectral resolution achievable with conventional flow cytometers is limited by the decrease in detection sensitivity which accompanies any reduction in the spectral range of the fluorescence detectors. This decrease in sensitivity is aggravated in systems with sequential optical filters, since losses increase for each additional detector. No one has published the achievement of an acceptable signal-to-noise ratio for a flow cytometer having more than five spectral channels.